Deterministic programming of human pluripotent stem cells into microglia facilitates studying their role in health and disease

Significance We here present a method for the manufacture of pure bulk quantities of microglia from human pluripotent stem cells (hPSCs) at unprecedented efficiency. We provide transcriptional, proteomic, and functional analysis of the microglia in two-dimensional (2D) cultures and single cell–resolution transcriptional profiling in 3D cortical organoids. This versatile technology of hPSC-derived microglia will improve in vitro models of the human brain and neurological disease. The platform will also facilitate biomedical research, including compound screening, drug discovery, and transplantation studies. We further demonstrate differential secondary MAPT genotype–dependent, microglial disease–associated phenotypes when microglia were placed in coculture with tau mutant cortical neurons. These findings provide evidence for mutation-dependent, differential pathophysiological effects on the immune response in the hereditary tauopathies.


Human iPSC lines and maintenance of pluripotency cultures
We used two independent, previously established, well-characterised, wild-type hiPSC lines: F13B hiPSCs were generated as part of the HipSci-project from adult skin fibroblasts by using Sendai virus reprogramming vectors at the Wellcome Trust Sanger Institute, United Kingdom (hPSCreg: WTSIi269-A) (1). C2-1 hiPSCs were generated from neonatal skin fibroblasts by using retrovirus vectors at the Max-Planck-Institute for Molecular Biomedicine, Germany (hPSCreg: MPIi001-A) (2). The study was approved by the local ethics committee (AZ 2019-390-f-S). All hiPSC lines were cultivated in a commercially available pluripotency maintaining medium (StemMACS iPS Brew XF, human; Miltenyi) in 6-well plates coated with growth-factor reduced Matrigel (Corning). The cells were kept in a humidified incubator at 37°C and 5% CO2. Fresh medium was added daily, and passaging was performed every 4-5 days using Accutase (Sigma).

Human ex vivo microglia
Human ex vivo microglia were obtained from the Netherlands Brain Bank (NBB). They stem from a non-demented female aged 60 years. Microglia were isolated post mortem, following the protocol by Mizee et al. (3). Briefly, autopsy was performed with approximately 6h post-mortem delay. Subcortical white matter and occipital cortex grey matter were mechanically dissected and enzymatically dissociated using collagenase I or trypsin for 60 min. Enzyme activity was quenched using fetal calf serum (FCS) and cell suspension was centrifuged. The cell pellet was resuspended in Dulbecco's Modified Eagle's Medium (DMEM) containing FCS and antibiotics and passed through a 100µm sieve. With Percoll gradient centrifugation microglia were finally isolated. After two washing steps with DMEM containing FCS and antibiotics, positive selection of microglia with anti-CD15 and subsequently with anti-CD11b magnetic microbeads was performed using magnetic activated cell sorting (Miltenyi Biotec). Isolated primary microglia were resuspended in TRIsure buffer and stored at -80°C. Subsequent RNA isolation was performed using phase separation by addition of chloroform and centrifugation and following RNA precipitation in isopropanol.

Molecular Cloning
The hROSA26 donor plasmid and the Cas9n and hROSA26 guide RNAs expressing plasmids were used as described previously (4,5). AAVS1 ZFN expression vectors were used from the same source (4,5). The AAVS1 donor vector was constructed by replacing EGFP with a bicistronic PU.1-T2A-C/EBPβ expression cassette. PU.1 was amplified by PCR from pWPT-hSPI1 which was a generous gift from Thomas Moreau (NHSBT Cambridge Centre). C/EBPβ was amplified by PCR from a C/EBPβ expression plasmid which was purchased from Dharmacon. During PCR amplification we inserted T2A and EcoRI/SpeI restriction sites. The vector backbone was restricted using EcoRI and SpeI to remove EGFP and all three fragments were purified by extraction from an agarose gel and were combined by Gibson Assembly (New England Biolabs). For the generation of the CLYBL-mCherry donor vector pC13N-iCAG.copGFP (Addgene: #66578; (6)) we first replaced the kanamycin resistance cassette by a hygromycin resistance cassette. Therefore, the vector was restricted with XmaJI and CsiI and hygromycin was PCR amplified from pSH231-EF1-RFP-HYGRO (Addgene: #115145; (6)). Both were purified by agarose gel extraction and ligated using Gibson Assembly. In a second step, copGFP was replaced by mCherry by cutting pC12N-iCAG.copGFP_hygro with Bsp1407 and MluI. MCherry was PCR-amplified from CLYBL_hOPM (Addgene: #112499). Both fragments were again purified by agarose gel extraction and ligated by Gibson assembly.

Nucleofection
Targeting of the human orthologue of the mouse ROSA26 locus (hROSA26) in hiPSCs was performed as we described recently (5). Briefly, a single cell suspension was generated by incubation of cells with Accutase for 5 min at 37°C. 2 x 10 6 cells were resuspended in 100µl nucleofection solution containing 4 µg of each plasmid (two plasmids encoding the hROSA26 guide RNAs and CAS9 nickase and the donor plasmid containing the CAG-rtTA transgene) and electroporated by using the program B-16 of the Amaxa nucleofector. After 5 min incubation time at room temperature (RT), 500µl of warm iPS medium containing Rho-associated protein kinase (ROCK) inhibitor Y-27632 (tebubio) was added into the cuvette. After another 5 min incubation time at RT nucleofected cells were plated on Matrigel-coated culture dishes (10 cm) in medium containing ROCK-inhibitor. 24h after nucleofection ROCK-inhibitor was removed and cells were cultured in iPS medium. After approximately 5 days, when non-confluent colonies had emerged, neomycin-resistant cells were selected by adding G418 (100µg/ml) for 5-7 days. G418-resistant colonies were individually picked, expanded and analyzed by genotyping. Established hROSA26_CAG-rtTA cell lines were targeted using the new AAVS1 donor plasmid with the inducible transgene cassette containing the two TFs (see above). Targeting was again performed by nucleofection, and antibiotic selection was performed using puromycin (0,3µg/ml). After colony picking, the clones were analyzed by genotyping and immunocytochemistry against the two inducible transgenes (PU.1 and C/EBPβ) after 24h doxycycline induction. For the generation of microglia reporter cell lines, established dual hROSA26/AAVS1 inducible cell lines were subjected to a third GSH targeting in the CLYBL locus. For this, we used a donor plasmid containing either copGFP or mCherry and a hygromycin resistance cassette. For site specific integration we used two TALEN plasmids (addgene #62197 & #62197; (6)). The three plasmids were nucleofected as described for the two previous GSH targeting steps. After transfection, antibiotic selection was carried out using hygromycin (50µg/ml). Picked clones were checked for homogenous reporter protein expression by fluorescence microscopy and flow cytometry.

Immunocytochemistry
Cells on coverslips or in a culture plate were fixed in 4% paraformaldehyde (PFA) for 15 min at RT and washed three times using DPBS [-]CaCl2 [-]MgCl2 (PBS-/-). Subsequently, cells were blocked with a blocking solution of PBS-/-containing 10% serum (Sigma Aldrich) and 0.3% Triton X-100 (Sigma Aldrich) for 30 min at RT. The cells were then incubated with a primary antibody solution containing 2% serum, 0.1% Triton X-100 and the appropriately diluted antibody over night at 4°C. After washing the cells three times with PBS-/-the secondary antibody solution, containing 1% serum, 0.1% Triton X-100 and the appropriately diluted secondary antibody and 4',6-diamidino-2phenylindole (DAPI, 1µg/ml, Thermo Fisher Scientific), for one hour at RT in the dark. After three washing steps using PBS-/-cells were analyzed using a fluorescence microscope (Leica, DMI6000 B).

Flow cytometry
For the flow cytometric analysis of surface marker expression, the cells were collected as single cell suspension and transferred through a cell strainer (40µm). Cells were washed in FACS buffer consisting of PBS-/-, 0.5% BSA and 2 mM EDTA and then incubated in FACS buffer containing the extracellular antibodies in an appropriate dilution for 15 min at RT in the dark. Cells were again washed in FACS buffer, resuspended in 100-200µl of FACS buffer and then analyzed on a Gallios Flow cytometer (Beckman Coulter).

Phagocytosis assay
a. Fluoresbrite microbead To investigate the phagocytic activity of MGLs we added fluorescently labelled latex beads in a ratio of 100 beads/cell. Analysis was performed after 1, 3, 5, and 24h of incubation. For flow cytometric analysis, we detached the cells after the respective incubation time, washed 3x with PBS-/-, resuspended the cells in FACS buffer and performed analysis using a Beckman Coulter Gallios Flow Cytometer. Additionally, cells were deposited on microscope slides using a cytospin. Cells were mounted with Fluoromount containing DAPI and imaged with an inverted microscope (BZ-9000 BioRevo; Keyence).
b. TAMRA labelled Amyloid-β Aβ1-42 (AnaSpec) was reconstituted to a stock solution of 1mg/ml in 1% NH4OH as stated in the data sheet, further diluted to 100µg/ml using endotoxin-free water. The solution was vortexed thoroughly and incubated at 37°C for 7days to form fibrils. Prior to cell exposure fAβ was thoroughly mixed. After 24h cells were collected, washed with PBS-/-and analyzed using flow cytometry. Another part of the cells was fixed in 4% PFA and counterstained with the microglia marker IBA1.

Cytokine secretion assay
For the investigation of cytokine secretion, MGLs, MACs and monocytes were stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 48h. The supernatants were collected, and the cytokine secretion assay was performed following manufacturer's instructions (LEGENDplex human macrophage/microglia panel, Biolegend).

ROS measurements and calcium imaging
For live cell imaging, an epifluorescence inverted microscope equipped with a 40x oil-immersion fluorite objective was used. Fluorescence data was analyzed with MetaFluor Fluorescence Ratio Imaging Software (Molecular Devices, LLC, Canada/US) and Origin, Version 2019 (OriginLab Corporation, Northampton, MA, USA). Dyes were diluted in artificial cerebrospinal fluid (120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 22 mM NaHCO3, 25 mM glucose, 2 mM CaCl2, 2 mM MgSO4). MGLs were plated on PLL (5 μg/ml) coated coverslips (Ø 12 mm). For detection of cytosolic ROS levels, cells were stained with 16 µM dihydroethidium (DHE, D11347, Invitrogen™) and measured immediately. Excitation light was provided by a LED lamp at 530 nm and emitted light was detected >670 nm. DHE fluorescence was measured with a frame interval of 5 seconds and fluorescence increase over time was calculated to determine the rate of ROS production. To evaluate cytosolic calcium levels, cells were preincubated for 30 min with 5 µM Fura-2 (AM, F1221, Invitrogen™). During the experiment, cells were kept in artificial cerebrospinal fluid. The ratiometric calcium indicator was excited at 340 nm and 380 nm and emitted light was detected at 510/80 nm. The frame interval was set to 1 second.

Seahorse -Oxygen Consumption
One day prior to seeding, the Seahorse XFp cell culture plates were coated with poly-D-lysine (PDL) [50μg/ml] and incubated at 37°C without CO2. Also, one day before the assay, sensor cartridges and surrounding chambers were hydrated with calibrant buffer (200 to 400 μl) and incubated overnight at 37°C without CO2. On the following day, plates were washed three times with 500 μl PBS, cells were seeded in NGD medium at a density of 1 x 10 5 cells per well and incubated for 3 days at 37°C. Each plate contained three wells of MGLs that were stimulated with lipopolysaccharides (LPS) (100ng/ml) for 24 h and three wells MGLs without LPS as a control group. Cells were measured in artificial CSF containing pyruvate (1 mM). After signal stabilization the cells were sequentially exposed to the mitochondrial stressors oligomycin (2 μM), FCCP (1 μM) and rotenone (0.5 μM), plus antimycin A (0.5 μM). OCR was determined using a Seahorse XFp Analyzer (Agilent), and assays have been analyzed with Wave Desktop software (Agilent).

Mitochondrial network analysis
To examine mitochondria in MGLs, cocultures of neurons and MGLs (plated on Matrigel approx. 4:1) were stained with 20 nM MitoTracker™ (M22426, Invitrogen™) for 30 min. Imaging was conducted with a Confocal Laser Scanning Microscope Leica SP8 equipped with a 63x oilimmersion objective. Reporter MGLs expressing mCherry were identified at 561 nm (emission >570 nm) and MitoTracker™ fluorescence was excited with a multiargon laser at 633 nm. Emitted light was gathered with a wavelength >670 nm and images were analyzed with ImageJ 1.52p (Wayne Rasband, National Institute of Health, USA) as described by Valente et al. (10). In short, fluorescent intensity threshold was manually set to allow optimal visualization of the mitochondrial network. MitoTracker fluorescence was transformed into a binary (black and white) output under visual control for adequate transformation. To further elucidate mitochondrial morphology, the binary was skeletonized, and single mitochondrial branch length was determined from this skeletonized mitochondrial network as described previously ((10); see Figure S16 for visual explanation of image processing). Average branch length of these single mitochondrial branches was determined as a surrogate marker for mitochondrial integrity and the degree of fusion and fission within the mitochondrial network.

Brain organoid generation & microglia-organoid coculture
Cerebral organoids were generated according to the original Lancaster protocol using the same wild-type hiPSC line used for the microglia derivation (11). 30 days old organoids were then cocultured with mature MGLs. For ICC, coculture organoids were fixed overnight in 4% PFA, embedded in Optimal cutting temperature compound (OCT) and frozen prior sectioning using a standard cryostat. 20-µm sections were used for immunostaining after 3 or 30 days in co-culture. For scRNA sequencing, 100 organoids were pooled and dissociated using Accutase, followed by FACS for viable cells (using a co-staining of Calcein AM (ThermoFisher, C1430), Zombie NIR (Biolegend, 423105) and DAPI) and subsequent RNA isolation.

PBMC and monocyte isolation from human blood and differentiation into macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood by density gradient centrifugation with lymphocyte separation medium. Subsequently monocytes were isolated with magnetic bead isolation following manufacturer's instructions (Monocyte Isolation Kit II (indirect, labelling of non-monocytes); Miltenyi Biotech). Monocytes were immediately used for downstream applications, plated on PLL-coated culture dishes and cultivated in NGD medium or differentiated into macrophages by either cultivating them in RPMI medium containing 10% FCS, 1% Pen/Strep and M-CSF (MAC) or in XVIVO-10 medium containing GM-CSF (100 ng/ml) for serum-free macrophage differentiation (MACsf). After 5 days, cells were either used for further experiments and analysis or cultured in NGD medium for another 72 h prior to analysis.

RNA isolation and quantitative RT-PCR (qPCR)
RNA was isolated using the Qiagen RNeasy Mini Kit and the On-column DNase I Digestion Set (Sigma-Aldrich). Subsequent cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). For qPCR the Maxima SYBR Green Master Mix was used (Thermo Fisher Scientific). qPCR reactions were run on a StepOne System (Applied Biosystems). All samples were analyzed in technical duplicates and normalized to the housekeeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH). Results were analyzed with the ΔΔCt method.

Proteome analysis
Normalization: Cells were lysed in 200 µl lysis buffer consisting of 200mM HEPPS in 8M urea, pH 8.5 supplemented with 1x Halt Protease and Phosphotase Inhibitor (Thermo Fisher). Protein lysates were sonicated for 20 sec on 10 % power. After centrifugation of the samples at 1.400 rpm for 30 min at 4°C, the supernatants were transferred to new tubes. Subsequently, protein concentrations were determined using a commercial BCA kit (Thermo Fisher). Protein adjusted sample aliquots were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fine-adjust protein amounts for label free proteome analysis. After staining the gel with Coomassie Blue according to manufacturer's protocol the optical density of each sample lane was determined with a calibrated gel scanner in transmission mode and the relative protein amount was calculated. Digestion and fractionation: Sample preparation for mass spectrometry was performed via insolution digestion and strong cation exchange (SCX) fractionation. In brief, samples were four-fold diluted in 25 mM NH4HCO3, pH 8.0, and subsequently incubated with 5 mM dithiothreitol at RT for 1 h. Afterwards, reduced cysteine residues were carbamidomethylated via addition of 20 mM iodine acetamide at RT for 1 h. Proteins were digested by adding 2.5 μg trypsin (TrypsinGold, Promega, Madison, WI, USA) and incubated at RT overnight. Digestion was stopped by adding formic acid (FAc) to a final concentration of 0.5% and subsequently centrifuged at 15.000 x g at 4°C for 15 min. Data processing: Raw data processing and protein identification of the high resolution orbitrap datasets were performed with de novo sequencing algorithms of PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Waterloo, Canada) using the SwissProt database. The false discovery rate was set to <1%.Raw data, complete identification listings and supplementary information are available via ProteomeExchange with identifier PXD024649.

GO-clustering and visualization:
The single enrichment analysis was performed by comparing the accumulated, annotated Gene Ontology terms (12) of the MGL proteome with those of a randomly generated, equal-sized human proteome. To calculate the significance of the difference between each proteome the fisher's exact was used and false positives were corrected using the Benjamini & Hochberg method (13). Utilizing the underlying structure of the Gene Ontology, these significant different terms were clustered based on their child-parent relationship, grouping significant child terms with their significant parent terms. Afterwards a graph was generated using the proteins as nodes that are connected by their significant parent terms by edges. This graph was then visualized with Gephi (http://gephi.org) using a force-directed graph drawing algorithm to calculate the layout, where virtual forces are applied to the nodes and edges. Whereas nodes generally push each other away, the edges pull them togetherthe more similar the proteins are to each other, the stronger the force of attraction. Therefore, defined by their shared GO terms, that are depicted as the connections (edges) in the graph, dense protein clusters are highly similar whereas dissimilar proteins show a spatial distance, because the number of shared terms (edges) are not sufficient to overcome the pushing forces of the proteins when the layout is calculated. In Figure S7, scatter plots are shown depicting the expression of pre-selected protein groups throughout all three biological replicates. For that purpose, downstream bioinformatic analysis was performed using Perseus v 1.6.14.0 as previously described (14,15). LFQ values were logarithmized (log2(x)) and missing values were replaced from normal distribution after deleting all proteins only detected in one out of three biological replicates. The data was searched against the integrated Perseus reference database for homo sapiens downloaded in March 2021 (http://annotations.perseus-framework.org).

Bulk RNA-sequencing
mRNA enrichment (NEB) was followed by a directional library preparation (NEB) and single-end sequencing on an Illumina NextSeq 500 instrument was performed at an average depth of 18.0 million reads. A total of 14772 expressed genes were detected per sample (range, 13017 -16911). Transcript-level expression was quantified from the raw reads (FASTQ format) using the Gencode v35 annotation and the Salmon software package (version 1.3.0). Downstream analyses were performed in the R environment (version 4.1.0). Gene-level summarization of counts and combination of all sample data was carried out with tximport (version 1.20.0) and the tximport list was transformed into a DESeqDataSet with DESeq2 (version 1.30.1). A gene with 10 or more counts was considered expressed. Raw counts were transformed using variance stabilizing transformations (VST) function of DESeq2. A total of 14611 expressed genes were detected per sample (range, 13017 -16911).

Integration with publicly available datasets
To compare different MGLs and effects of in vitro culture, we integrated publicly available datasets from three differentiation protocols (7,16,17) (GSE85839, n=16). Moreover, we included RNA expression profiles from an extensive dataset of primary human microglia in vitro and ex vivo, and bloodisolated monocytes and macrophages (18) (Table S2 from (18), n=64). Processed RNA-seq data was normalized using the removeBatchEffect() function of the limma package (v3.5) specifying the datasets as batch. Normalized data were log transformed and a matrix with the 1,000 most variably expressed genes across all datasets was used as input to generate the MDS analysis using the plotMDS() function of the limma package.

Generation of single cell RNA libraries and sequencing
The samples were loaded onto the 10x Genomics Chromium Single Cell Controller with the Chromium Single Cell 3' Library and Gel Beads Kit v3. Library preparation was carried out according to the manufacturer's instruction using AMPure beads (Beckman Coulter). Sequencing was performed on an Illumina Nextseq 6000 with a 150-8-0-150 read setup. The bcl files were demultiplexed with cellranger mkfastq v3.1 and counted with cellranger count v3.1 according to the manufacturer's instructions.

Single cell RNA sequencing data analysis
Downstream analysis was performed with R v4.1 and Seurat v4 (19) based on the official vignettes. Briefly, low quality cells were removed by filtering cells with few genes (<200) or high genes (>7000) or high mitochondrial percentages (15%) for each sample individually. The data were normalized using logarithmic transformation normalization, highly variable genes were identified, and the data were scaled regressing out mitochondrial percentage and sequencing depth. Principal component analysis was performed and the first 30 PCs were used for Uniform Manifold Approximation and Projection (UMAP). The clusters were determined in Seurat with the FindNeighbors and FindClusters function and annotated based on known marker genes. The steps were reperformed for the microglia subset. The top markers were identified with the FindMarkers function in Seurat based on the Wilcoxon rank sum test and an adjusted p value threshold (Bonferroni correction) of 0.05 and average log2 fold change of 0.25. Volcano plots were produced with Enhanced Volcano v1.10. The gene set scores were calculated with the AddModuleScore function in Seurat. Interactions between the neuroectoderm and microglia cluster were identified with CellPhoneDB v2.1.7 (20) following the official instructions. Statistical iterations were set at 1000 and the threshold of the cells expressing the ligand/receptor was set at 10%.

B. Materials
Dataset S1. Marker genes of clusters in the organoid-microglia coculture (corresponding to